Attenuated Salmonella Typhimurium with truncated LPS and outer membrane-displayed RGD peptide for cancer therapy.

Gram-negative, facultatively anaerobic bacteria Salmonella Typhimurium is a candidate agent or delivery vector for cancer therapy. Effective targeted therapies in addition to radiotherapy, chemotherapy and surgery have been urgently needed as an alternative or supplement. This study expected to further improve the tumor-targeting ability of Salmonella bacteria through genetic modifications. Based on an auxotrophic Salmonella bacterial strain (D2), we constructed Salmonella mutants with altered LPS length to facilitate displaying the RGD4C targeting peptide on the outer membrane surface of Salmonella. The expression of RGD4C peptide in fusion with OmpA was identified by outer membrane protein extraction and WB detection in different mutant strains. However, flow cytometry analysis following immunofluorescence staining demonstrated that the extracellular length of Salmonella LPS did affect the surface display of RGD4C peptide. The strain D2-RGD4C that synthesized intact LPS including lipid A, core oligosaccharides and O antigen polysaccharides could hardly display RGD4C peptide, showing the same fluorescence signal intensity as the strains not expressing RGD4C peptide. Among different strains, D2 ∆rfaJ-RGD4C that synthesized truncated LPS including lipid A and partial core oligosaccharides was capable of displaying RGD4C peptide most efficiently and showed the highest ability to target HUVECs expressing αV integrin and tumor tissue with abundant neovascularization. Animal experiments also demonstrated that this tumor-targeting attenuated Salmonella strain to simultaneously deliver endostatin and TRAIL, two agents with different anti-tumor activities, could significantly inhibit tumor growth and prolong mouse survival. Thus, our studies revealed that Salmonella could be genetically engineered to improve its tumor targeting via the truncation of LPS and surface display of targeting peptides, thereby eliciting superior anti-tumor effects through targeted delivery of drug molecules.

Mycobacterium tuberculosis PE17 (Rv1646) promotes host cell apoptosis via host chromatin remodeling mediated by reduced H3K9me3 occupancy.

Tuberculosis caused by Mycobacterium tuberculosis remains a serious global public health threat. M. tuberculosis PE and PPE proteins are closely involved in pathogen-host interaction. To explore the predicted function of the M. tuberculosis PE17 (Rv1646), we heterologously expressed PE17 in a non-pathogenic Mycobacterium smegmatis strain (Ms_PE17). PE17 can reduce the survival of M. smegmatis within macrophages associated with altering the transcription levels of inflammatory cytokines IL1ß, IL6, TNFα, and IL10 in Ms_PE17 infected macrophages through JNK signaling. Furthermore, macrophages apoptosis was increased upon Ms_PE17 infection in a caspases-dependent manner, accompanied by the activation of the Endoplasmic Reticulum stress IRE1α/ASK1/JNK signaling pathway. This can be largely interpreted by the epigenetic changes through reduced H3K9me3 chromatin occupancy post Ms_PE17 infection. To our knowledge, this is the first report that PE17 altered the macrophages apoptosis via H3K9me3 mediated chromatin remodeling.

Mycobacterium tuberculosis Rv0580c Impedes the Intracellular Survival of Recombinant Mycobacteria, Manipulates the Cytokines, and Induces ER Stress and Apoptosis in Host Macrophages via NF-κB and p38/JNK Signaling.

The Mycobacterium tuberculosis (M. tb) genome encodes a large number of hypothetical proteins, which need to investigate their role in physiology, virulence, pathogenesis, and host interaction. To explore the role of hypothetical protein Rv0580c, we constructed the recombinant Mycobacterium smegmatis (M. smegmatis) strain, which expressed the Rv0580c protein heterologously. We observed that Rv0580c expressing M. smegmatis strain (Ms_Rv0580c) altered the colony morphology and increased the cell wall permeability, leading to this recombinant strain becoming susceptible to acidic stress, oxidative stress, cell wall-perturbing stress, and multiple antibiotics. The intracellular survival of Ms_Rv0580c was reduced in THP-1 macrophages. Ms_Rv0580c up-regulated the IFN-γ expression via NF-κB and JNK signaling, and down-regulated IL-10 expression via NF-κB signaling in THP-1 macrophages as compared to control. Moreover, Ms_Rv0580c up-regulated the expression of HIF-1α and ER stress marker genes via the NF-κB/JNK axis and JNK/p38 axis, respectively, and boosted the mitochondria-independent apoptosis in macrophages, which might be lead to eliminate the intracellular bacilli. This study explores the crucial role of Rv0580c protein in the physiology and novel host-pathogen interactions of mycobacteria.

Optimized Attenuated Salmonella Typhimurium Suppressed Tumor Growth and Improved Survival in Mice.

The gram-negative facultative anaerobic bacteria Salmonella enterica serovar Typhimurium (hereafter S. Typhimurium) has always been considered as one candidate of anti-tumor agents or vectors for delivering drug molecules. In this study, we compared several widely studied S. Typhimurium strains in their anti-tumor properties aiming to screen out the best one for further optimization and use in cancer therapy. In terms of the motility, virulence and anti-tumor efficacy, the three strains 14028, SL1344, and UK-1 were similar and obviously better than LT-2, and UK-1 showed the best phenotypes among them. Therefore, the strain UK-1 (D) was selected for the following studies. Its auxotrophic mutant strain (D1) harboring ∆aroA and ∆purM mutations was further optimized through the modification of lipid A structure, generating a new strain named D2 with stronger immunostimulatory activity. Finally, the ∆asd derivative of D2 was utilized as one live vector to deliver anti-tumor molecules including the angiogenesis inhibitor endostatin and apoptosis inducer TRAIL and the therapeutic and toxic-side effects were evaluated in mouse models of colon carcinoma and melanoma. After intraperitoneal infection, engineered Salmonella bacteria equipped with endostatin and/or TRAIL significantly suppressed the tumor growth and prolonged survival of tumor-bearing mice compared to PBS or bacteria carrying the empty plasmid. Consistently, immunohistochemical studies confirmed the colonization of Salmonella bacteria and the expression of anti-tumor molecules inside tumor tissue, which were accompanied by the increase of cell apoptosis and suppression of tumor angiogenesis. These results demonstrated that the beneficial anti-tumor efficacy of attenuated S. Typhimurium bacteria could be improved through delivery of drug molecules with powerful anti-tumor activities.

Human gene expression profiling identifies key therapeutic targets in tuberculosis infection: A systematic network meta-analysis.

Tuberculosis (TB) is one of the deadliest diseases since ancient times and is still a global health problem. So, there is a need to develop new approaches for early detection of TB and understand the host-pathogen relationship. In the present study, we have analyzed microarray data sets and compared the transcriptome profiling of the healthy individual with latent infection (LTBI) and active TB (TB) patients, and identified the differentially expressed genes (DEGs). Next, we performed the systematic network meta-analysis of the DEGs, which identified the seven most influencing hub genes (IL6, IL1B, TNF, NFKB1, STAT1, JAK2, and MAPK8) as the potential therapeutic target in the tuberculosis disease. These target genes are involved in many biological processes like cell cycle control, apoptosis, complement signalling, enhanced cytokine & chemokine signalling, pro-inflammatory responses, and host immune responses. Additionally, we also identified 22 inferred genes that are mainly engaged in the induction of innate immune response, cellular response to interleukin-6, inflammatory response, apoptotic process, I-kappaB-phosphorylation, JAK-STAT signalling pathway, macrophage activation, cell growth, and cell signalling. The proper attention of these inferred genes may open up a new horizon to understand the defensive mechanisms of TB disease. The transcriptome profiling and network approach can enhance the understanding of the molecular pathogenesis of tuberculosis infection and have implications for the plan and execution of mRNA expression tools to support early diagnostics and treatment of Mycobacterium tuberculosis (M.tb).

Bacteria-derived minicells for cancer therapy.

Bacteria are always a considerable tool for the cancer therapy. The bacteria-derived minicells are re-emerged as a promising drug delivery system for cancer therapy. The minicells are nano-sized, anucleated, non-dividing, and metabolically active cells produced by abnormal bacterial cell division that are able to transcribe and translate the gene of interest. Minicells encapsulate a wide range of chemotherapeutic and molecular drugs, si/shRNA, antigens and therapeutic toxins to precisely deliver them to the cancer cells through the easy modification of minicell surface with bi-specific antibodies against receptor-targeted cancer cells. Minicell-mediated chemotherapy may inhibit the growth of drug-resistant tumors and exhibits the potential to successfully deliver the chemotherapeutics into hypoxic and necrotic region in solid tumors. This novel approach significantly overcomes drug leakage and severe toxicity by enhancing targeting specificity and therapeutic index of drugs in cancer therapy. Many antibody-conjugated drug-loaded minicells are being investigated in clinical trials for cancer therapy. This review summarizes the advantages of bacteria-derived minicells as delivery systems for anti-cancer drugs or agents and the recent advances and emerging future in cancer therapy.

Mycobacterium smegmatis msmeg_3314 is involved in pyrazinamide and fluoroquinolones susceptibility via NAD+/NADH dysregulation.

Aim: To identify and characterize new mycobacterium pyrazinamide (PZA) resistance genes in addition to pncA, rpsA and panD. Materials & methods: To screen a Tn7 M. smegmatis mc2155 transposon library using 50 µM PZA and a PZA hypersensitive mutant (M492) was obtained. MIC was further used to confirm the hypersensitivity of M492 mutant by culturing the mutant in Middlebrook 7H9 liquid medium at 37°C. Results:msmeg_3314 is the gene underlying the hypersensitive phenotype of mutant M492. The observed resistance to PZA and fluoroquinolones involved the alteration of Mycobacterium cell wall permeability and the dissipation of the proton motive force. NAD+/NADH dysregulation and attenuated glyoxylate shunt might underlie the declined scavenging capacity of reactive oxygen species in the msmeg_3314-deficient mutants. Conclusion:msmeg_ 3314 is a novel gene involved in pyrazinamide resistance and might be a new candidate for drugs target.

Mycobacterium tuberculosis PE31 (Rv3477) Attenuates Host Cell Apoptosis and Promotes Recombinant M. smegmatis Intracellular Survival via Up-regulating GTPase Guanylate Binding Protein-1.

The Mycobacterium (M.) tuberculosis comprising proline-glutamic acid (PE) subfamily proteins associate with virulence, pathogenesis, and host-immune modulations. While the functions of most of this family members are not yet explored. Here, we explore the functions of "PE only" subfamily member PE31 (Rv3477) in virulence and host-pathogen interactions. We have expressed the M. tuberculosis PE31 in non-pathogenic Mycobacterium smegmatis strain (Ms_PE31) and demonstrated that PE31 significantly altered the cell facet features including colony morphology and biofilm formation. PE31 expressing M. smegmatis showed more resistant to the low pH, diamide, H2O2 and surface stress. Moreover, Ms_PE31 showed higher intracellular survival in macrophage THP-1 cells. Ms_PE31 significantly down-regulated the production of IL-12p40 and IL-6, while up-regulates the production of IL-10 in macrophages. Ms_PE31 also induced the expression of guanylate-binding protein-1 (GBP-1) in macrophages. Further analysis demonstrates that Ms_PE31 inhibits the caspase-3 activation and reduces the macrophages apoptosis. Besides, the NF-κB signaling pathway involves the interplay between Ms_PE31 and macrophages. Collectively, our finding identified that PE31 act as a functionally relevant virulence factor of M. tuberculosis.

Mycobacterium tuberculosis metC (Rv3340) derived hydrogen sulphide conferring bacteria stress survival.

Tuberculosis, especially multidrug resistant cases, remains an enormous public health threat. Mycobacterium tuberculosis metC (Rv3340) an enzyme involved in methionine biosynthesis was identified and characterised for antimicrobial susceptibility. We reported that the overexpression of Rv3340 in Mycobacterium smegmatis (Ms_Rv3340) produces hydrogen sulphide (H2S) for its energy in harsh conditions. The produced H2S sustained Ms_Rv3340 against streptomycin, whereas the chemical inhibition of H2S caused streptomycin lethality to Ms_Rv3340. Further analysis showed that cysteine-H2O2 treatment of Ms-Rv3340 initiated DNA damage via Fenton reaction. Ms_Rv3340 downregulated the expression levels of three streptomycin responsive genes. To our knowledge, no study has been previously reported that M. tuberculosis metC (Rv3340) can generates H2S modulating resistant to streptomycin which provides a greater perception toward the treatment and control of tuberculosis.

Regulation of host cell pyroptosis and cytokines production by Mycobacterium tuberculosis effector PPE60 requires LUBAC mediated NF-κB signaling.

Tuberculosis, caused by Mycobacterium tuberculosis infection, remains a global public health threat. The success of M. tuberculosis largely contributes to its manipulation of host cell fate. The role of M. tuberculosis PE/PPE family effectors in the host destiny was intensively explored. In this study, the role of PPE60 (Rv3478) was characterized by using Rv3478 recombinant M. smegmatis. PPE60 can promote host cell pyroptosis via caspases/NLRP3/gasdermin. The production of pro-inflammatory cytokines, such as IL-1ß, IL-6, IL-12p40 and TNF-α was altered by PPE60. We found that LUBAC was involved in PPE60-elicited NF-κB signaling by using Linear Ubiquitin Chain Assembly Complex (LUBAC)-specific inhibitor gliotoxin. The PPE60 recombinant M. smegmatis survival rate within macrophages is increased, as well as elevated resistance to stresses such as low pH, surface stresses and antibiotics exposure. For a first time it is firstly reported that M. tuberculosis effector PPE60 can modulate the host cell fate via LUBAC-mediated NF-κB signaling.

Mycobacterium tuberculosis PE_PGRS18 enhances the intracellular survival of M. smegmatis via altering host macrophage cytokine profiling and attenuating the cell apoptosis.

Mycobacterium tuberculosis PE/PPE family proteins, named after the presence of conserved PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains at N-terminal, are prevalent in M. tuberculosis genome. The function of most PE/PPE family proteins remains elusive. To characterize the function of PE_PGRS18, the encoding gene was heterologously expressed in M. smegmatis, a nonpathogenic mycobacterium. The recombinant PE_PGRS18 is cell wall associated. M. smegmatis PE_PGRS18 recombinant showed differential response to stresses and altered the production of host cytokines IL-6, IL-1ß, IL-12p40 and IL-10, as well as enhanced survival within macrophages largely via attenuating the apoptosis of macrophages. In summary, the study firstly unveiled the role of PE_PGRS18 in physiology and pathogenesis of mycobacterium.